Primary Endosymbiont Research Articles

Effects of host plants on aphid feeding behavior, fitness, and Buchnera aphidicola titer.

Aphids are sap-feeding plant pests that depend on their symbiotic relationships with the primary endosymbiont Buchnera aphidicola to adapt to impoverished diets. However, how the host plant affects the aphid primary symbiont and aphid adaptation to host plant transfer are poorly known. In this study, aphid symbiont screening and genotype identification were used to establish 2 aphid strains (Rhopalosiphum maidis [Rm] and Rhopalosiphum padi [Rp] strains) containing only Buchnera without any secondary symbionts for both wheat aphid species (R. maidis and R. padi). Aphid fitness and Buchnera titers were unstable on some of these host plants after transferring to novel host plants (G1-G5), which were influenced by host plant species and generations; however, they stabilized after prolonged feeding on the same plants for 10 generations. The electropenetrography (EPG) records showed that the allocation of aphid feeding time was significantly distinct in the 6 host plants; aphids had more intracellular punctures and spent more nonprobing time on green bristlegrass which was not conducive to its growth compared with other plants. The content of soluble sugar, soluble protein, and amino acid in the leaves of the 6 host plants were also clearly separated. The correlation coefficient analysis showed that the nutrient contents of host plants had significant correlations with aphid feeding behaviors, fitness, and Buchnera titers. In the meantime, aphid fitness, and Buchnera titers were also affected by aphid feeding behaviors. Also, Buchnera titers of aphid natural populations on 6 host plants showed a visible difference. Our study deepened our understanding of the interaction among aphids, endosymbionts, and host plants, indicating that the host plant nutrient content is a predominant factor affecting aphid adaptation to their diet, initially affecting aphid feeding behaviors, and further affecting aphid fitness and Buchnera titers, which would further contribute to exploiting new available strategies for aphid control.

'Drifting' Buchnera genomes track the microevolutionary trajectories of their aphid hosts.

Evolution of Buchnera-aphid host symbioses is often studied among species at macroevolutionary scales. Investigations within species offer a different perspective about how eco-evolutionary processes shape patterns of genetic variation at microevolutionary scales. Our study leverages new and publicly available whole-genome sequencing data to study Buchnera-aphid host evolution in Myzus persicae, the peach potato aphid, a globally invasive and polyphagous pest. Across 43 different asexual, clonally reproducing isofemale strains, we examined patterns of genomic covariation between Buchnera and their aphid host and considered the distribution of mutations in protein-coding regions of the Buchnera genome. We found Buchnera polymorphisms within aphid strains, suggesting the presence of genetically different Buchnera strains within the same clonal lineage. Genetic distance between pairs of Buchnera samples was positively correlated to genetic distance between their aphid hosts, indicating shared evolutionary histories. However, there was no segregation of genetic variation for both M. persicae and Buchnera with plant host (Brassicaceae and non-tobacco Solanaceae) and no associations between genetic and geographic distance at global or regional spatial scales. Abundance patterns of non-synonymous mutations were similar to synonymous mutations in the Buchnera genome, and both mutation classes had similar site frequency spectra. We hypothesize that a predominance of neutral processes results in the Buchnera of M. persicae to simply 'drift' with the evolutionary trajectory of their aphid hosts. Our study presents a unique microevolutionary characterization of Buchnera-aphid host genomic covariation across multiple aphid clones. This provides a new perspective on the eco-evolutionary processes generating and maintaining polymorphisms in a major pest aphid species and its obligate primary endosymbiont.

Comparative genomics of the primary endosymbiont Buchnera aphidicola in aphid hosts and their coevolutionary relationships

BackgroundCoevolution between modern aphids and their primary obligate, bacterial endosymbiont, Buchnera aphidicola, has been previously reported at different classification levels based on molecular phylogenetic analyses. However, the Buchnera genome remains poorly understood within the Rhus gall aphids.ResultsWe assembled the complete genome of the endosymbiont Buchnera in 16 aphid samples, representing 13 species in all six genera of Rhus gall aphids by shotgun genome skimming method. We compared the newly assembled genomes with those from GenBank to comprehensively investigate patterns of coevolution between the bacteria Buchnera and their aphid hosts. Buchnera genomes were mostly collinear, and the pan-genome contained 684 genes, in which the core genome contained 256 genes with some lineages having large numbers of tandem gene duplications. There has been substantial gene-loss in each Buchnera lineage. We also reconstructed the phylogeny for Buchnera and their host aphids, respectively, using 72 complete genomes of Buchnera, along with the complete mitochondrial genomes and three nuclear genes of 31 corresponding host aphid accessions. The cophylogenetic test demonstrated significant coevolution between these two partner groups at individual, species, generic, and tribal levels.ConclusionsBuchnera exhibits very high levels of genomic sequence divergence but relative stability in gene order. The relationship between the symbionts Buchnera and its aphid hosts shows a significant coevolutionary pattern and supports complexity of the obligate symbiotic relationship.

Barley Yellow Dwarf Virus Influences Its Vector's Endosymbionts but Not Its Thermotolerance.

The barley yellow dwarf virus (BYDV) of cereals is thought to substantially increase the high-temperature tolerance of its aphid vector, Rhopalosiphum padi, which may enhance its transmission efficiency. This is based on experiments with North American strains of BYDV and R. padi. Here, we independently test these by measuring the temperature tolerance, via Critical Thermal Maximum (CTmax) and knockdown time, of Australian R. padi infected with a local BYDV isolate. We further consider the interaction between BYDV transmission, the primary endosymbiont of R. padi (Buchnera aphidicola), and a transinfected secondary endosymbiont (Rickettsiella viridis) which reduces the thermotolerance of other aphid species. We failed to find an increase in tolerance to high temperatures in BYDV-infected aphids or an impact of Rickettsiella on thermotolerance. However, BYDV interacted with R. padi endosymbionts in unexpected ways, suppressing the density of Buchnera and Rickettsiella. BYDV density was also fourfold higher in Rickettsiella-infected aphids. Our findings indicate that BYDV does not necessarily increase the temperature tolerance of the aphid transmission vector to increase its transmission potential, at least for the genotype combinations tested here. The interactions between BYDV and Rickettsiella suggest new ways in which aphid endosymbionts may influence how BYDV spreads, which needs further testing in a field context.

Effect of tree height and spraying methods on Diaphorina citri kuwayama endosymbionts in the context of Huanglongbing disease management in citrus orchards.

Huanglongbing (HLB) (caused by Candidatus Liberibacter asiaticus) is the most damaging disease of citrus around the world. This study investigated the effects of citrus tree height on Diaphorina citri Kuwayama mortality, endosymbiont responses, and HLB distribution. The results reveal that the age of citrus trees plays a significant role in psyllid mortality. Interestingly, the cumulative mean mortality (%) of psyllids over the seven-day observation period was higher (31.50±0.03) when four-year-old (501A1, 502A2, 501A3) citrus trees were sprayed with a US-SMART mechanical sprayer. In contrast, the psyllids mortality was 0.09±0.23 for the 13-year-old citrus trees (104A2, 104A3, 104C1) sprayed with a US-SMART mechanical sprayer and 9.10±0.05 for 13-year-old (502A2, 502B2, 502D1) citrus trees sprayed with a fixed US-SMART mechanical sprayer. Our findings also revealed that psyllids from both four- and 13-year-old citrus trees carried Candidatus Carsonella ruddii species and Wolbachia, the primary and secondary endosymbionts, respectively. Surprisingly, infection rates of these endosymbionts remained consistent across different age groups, as confirmed by quantitative polymerase chain reaction analysis. Furthermore, our study highlights the significance of tree height as a proxy for tree age in influencing HLB occurrence. Specifically, four-year-old citrus trees subjected to the US-SMART mechanical sprayer for citrus psyllid control demonstrated effective disease management compared to 13-year-old (104A2, 104A3, 104C1) citrus trees sprayed with US-SMART mechanicalsprayers. Additionally, the investigation explored the impact of tree height on HLB distribution. In four-year-old trees, no significant correlation between HLB disease and tree height was observed, potentially due to effective spray coverage with US-SMART mechanical sprayer. However, in 13-year-old (104A2, 104A3, 104C1) citrus tree sprayed with US-SMART mechanical sprayer, a positive correlation between tree height and HLB disease was evident. This research provides valuable insights into the complex interaction between citrus tree age, psyllid endosymbionts responses, and HLB distribution. These results emphasize effective HLB management strategies, especially in orchards with diverse tree age populations, ultimately contributing to the long-term sustainability of citrus cultivation. © 2023 Society of Chemical Industry.

Ecological and evolutionary inferences from aphid microbiome analyses depend on methods and experimental design

AbstractIntroductionAphids play an important role in agroecological contexts as pests and vectors of plant diseases. Aphid performance is closely connected to microbial endosymbionts that provide different benefits or costs to both the aphids and their hosts plants. Furthermore, the microbiome of aphids is connected to soil microbiomes via the plant. Aphid microbiome experiments usually include a pooling step, where several individuals are sequenced together to obtain sufficient DNA concentrations but pooling may blur intraspecific variations.Materials and MethodsTo investigate the effects of sequencing single versus pooled aphids on the results of microbiome analyses, we compared 16S rRNA/ITS amplicon libraries from pooled and single oak aphids (Tuberculatus annulatus HARTIG) under three different soil treatments. We tested whether results quantitatively or qualitatively depend on pooling aphids, prevalence‐based in silico filtering or removal of the primary endosymbiont (Buchnera aphidicola). Buchnera phylogeny, prevalence and abundance of secondary endosymbionts and effects of soil microbiota were investigated.ResultsPooling leads to quantitative differences in bacteria and qualitative differences in fungal species richness, bacterial community composition and partially fungal community composition. Filtering‐dependent results were obtained for bacterial evenness. Buchnera phylogeny supports the hypothesis of cospeciation of primary endosymbionts in oak aphids. We detected Arsenophonus, Hamiltonella, Rickettsia, Rickettsiella, Serratia and Sphingopyxis in oak aphids, with their prevalence and abundance partially affected by pooling. Pooling leads to overestimating the frequency of multispecies endosymbiont infections, while underestimating their relative abundance.ConclusionWe hereby extend our view on non‐model aphid microbiomes and identify pitfalls in experimental design in aphid microbiome research.

Division of labor within psyllids: metagenomics reveals an ancient dual endosymbiosis with metabolic complementarity in the genus Cacopsylla.

Heritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects by enabling the exploitation of nutritionally limited food sources. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacopsylla, consisting of the primary endosymbiont "Candidatus Carsonella ruddii" and a co-occurring Enterobacteriaceae bacterium for which we propose the name "Candidatus Psyllophila symbiotica." Its localization within the bacteriome and its small genome size confirm that Psyllophila is a co-primary endosymbiont widespread within the genus Cacopsylla. Despite its highly eroded genome, Psyllophila perfectly complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as Carsonella's, suggesting an ancient dual endosymbiosis that has now reached a precarious stage where any additional gene loss would make the system collapse. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.

Comparative evolutionary analyses of eight whitefly Bemisia tabaci sensu lato genomes: cryptic species, agricultural pests and plant-virus vectors

BackgroundThe group of > 40 cryptic whitefly species called Bemisia tabaci sensu lato are amongst the world’s worst agricultural pests and plant-virus vectors. Outbreaks of B. tabaci s.l. and the associated plant-virus diseases continue to contribute to global food insecurity and social instability, particularly in sub-Saharan Africa and Asia. Published B. tabaci s.l. genomes have limited use for studying African cassava B. tabaci SSA1 species, due to the high genetic divergences between them. Genomic annotations presented here were performed using the ‘Ensembl gene annotation system’, to ensure that comparative analyses and conclusions reflect biological differences, as opposed to arising from different methodologies underpinning transcript model identification.ResultsWe present here six new B. tabaci s.l. genomes from Africa and Asia, and two re-annotated previously published genomes, to provide evolutionary insights into these globally distributed pests. Genome sizes ranged between 616—658 Mb and exhibited some of the highest coverage of transposable elements reported within Arthropoda. Many fewer total protein coding genes (PCG) were recovered compared to the previously published B. tabaci s.l. genomes and structural annotations generated via the uniform methodology strongly supported a repertoire of between 12.8—13.2 × 103 PCG. An integrative systematics approach incorporating phylogenomic analysis of nuclear and mitochondrial markers supported a monophyletic Aleyrodidae and the basal positioning of B. tabaci Uganda-1 to the sub-Saharan group of species. Reciprocal cross-mating data and the co-cladogenesis pattern of the primary obligate endosymbiont ‘Candidatus Portiera aleyrodidarum’ from 11 Bemisia genomes further supported the phylogenetic reconstruction to show that African cassava B. tabaci populations consist of just three biological species. We include comparative analyses of gene families related to detoxification, sugar metabolism, vector competency and evaluate the presence and function of horizontally transferred genes, essential for understanding the evolution and unique biology of constituent B. tabaci. s.l species.ConclusionsThese genomic resources have provided new and critical insights into the genetics underlying B. tabaci s.l. biology. They also provide a rich foundation for post-genomic research, including the selection of candidate gene-targets for innovative whitefly and virus-control strategies.

Effects of fungicides on fitness and Buchnera endosymbiont density in Aphis gossypii.

Several agricultural fungicides are known to affect insect pests directly and these effects may be transgenerational and mediated through impacts on endosymbionts, providing opportunities for pest control. The cotton aphid Aphis gossypii is a polyphagous pest that can cause large crop yield losses. Here, we tested the effects of three fungicides, pyraclostrobin, trifloxystrobin and chlorothalonil, on the fitness and Buchnera endosymbiont of A. gossypii. The formulations of trifloxystrobin and pyraclostrobin, and the active ingredient of pyraclostrobin produced dose-dependent mortality in A. gossypii, whereas there was no dose-dependent mortality for chlorothalonil. The formulations of trifloxystrobin and pyraclostrobin significantly reduced the lifespan and fecundity of A. gossypii, and increased the density of Buchnera in the parental generation but not the (unexposed) F1 . When the active ingredient of pyraclostrobin was tested, the lifespan of the F0 generation was also reduced, but the density of Buchnera was not, indicating that non-insecticidal chemicals in the fungicide formulation may affect the density of the endosymbiont of A. gossypii. There was no transgenerational effect of the active ingredient of pyraclostrobin on the lifespan and Buchnera of (unexposed) F1 . Our results suggest that formulations of two strobilurin fungicides have immediate impacts on the fitness of A. gossypii, and chemicals in the formulation impact the density of the primary Buchnera endosymbiont. Our study highlights the potential effects of non-insecticidal chemicals of fungicides on aphid pests and their primary endosymbionts but direct connections between fitness and Buchnera densities remain unclear. © 2023 Society of Chemical Industry.

Insect phylogeny structures the bacterial communities in the microbiome of psyllids (Hemiptera: Psylloidea) in Aotearoa New Zealand.

The bacterial microbiome of psyllids has been studied for decades, with a strong focus on the primary and secondary endosymbionts capable of providing essential amino acids for the insects' diet and therefore playing a key role in the insects' ability to radiate on novel plant hosts. Here, we combine metabarcoding analysis of the bacterial communities hosted by psyllids with a multi-gene phylogenetic analysis of the insect hosts to determine what factors influence the bacterial diversity of the psyllids' microbiomes, especially in the context of the dispersal and evolutionary radiation of these insects in Aotearoa New Zealand. Using multi-gene phylogenetics with COI, 18S and EF-1α sequences from 102 psyllid species, we confirmed for the first time monophyly for all the six genera of native/endemic Aotearoa New Zealand psyllids, with indications that they derive from at least six dispersal events to the country. This also revealed that, after its ancestral arrival, the genus Powellia has radiated onto a larger and more diverse range of plants than either Psylla or Ctenarytaina, which is uncommon amongst monophyletic psyllids globally. DNA metabarcoding of the bacterial 16S gene here represents the largest dataset analysed to date from psyllids, including 246 individuals from 73 species. This provides novel evidence that bacterial diversity across psyllid species is strongly associated with psyllid phylogenetic structure, and to a lesser degree to their host plant association and geographic distribution. Furthermore, while the strongest co-phylogenetic signals were derived from the primary and secondary symbionts, a signal of phylosymbiosis was still retained among the remaining taxa of the bacterial microbiome, suggesting potential vertical transmission of bacterial lineages previously unknown to have symbiotic roles.

Diversity of endosymbionts in camellia spiny whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), estimated by 16S rRNA analysis and their biological implications.

Camellia spiny whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), is a major pest in tea, which poses a serious threat to tea production. Similar to many insects, various bacterial symbioses inside A. camelliae may participate in the reproduction, metabolism, and detoxification of the host. However, few reports included research on the microbial composition and influence on A. camelliae growth. We first applied high-throughput sequencing of the V4 region in the 16S rRNA of symbiotic bacteria to study its component and effect on the biological trait of A. camelliae by comparing it with the antibiotic treatment group. The population parameters, survival rate, and fecundity rate of A. camelliae were also analyzed using the age-stage two-sex life table. Our results demonstrated that phylum Proteobacteria (higher than 96.15%) dominated the whole life cycle of A. camelliae. It unveiled the presence of Candidatus Portiera (primary endosymbiont) (67.15-73.33%), Arsenophonus (5.58-22.89%), Wolbachia (4.53-11.58%), Rickettsia (0.75-2.59%), and Pseudomonas (0.99-1.88%) genus. Antibiotic treatment caused a significant decrease in the endosymbiont, which negatively affected the host's biological properties and life process. For example, 1.5% rifampicin treatment caused a longer preadult stage in the offspring generation (55.92 d) compared to the control (49.75d) and a lower survival rate (0.36) than the control (0.60). The decreased intrinsic rate of increase (r), net reproductive rate (R 0), and prolonged mean generation time (T) were signs of all disadvantageous effects associated with symbiotic reduction. Our findings confirmed the composition and richness of symbiotic bacteria in larva and adult of A. camelliae by an Illumina NovaSeq 6000 analysis and their influence on the development of the host by demographic research. Together, the results suggested that symbiotic bacteria play an important role in manipulating the biological development of their hosts, which might help us for developing new pest control agents and technologies for better management of A. camelliae.

Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen.

Sequencing high molecular weight (HMW) DNA with long-read and linked-read technologies has promoted a major increase in more complete genome sequences for nonmodel organisms. Sequencing approaches that rely on HMW DNA have been limited to larger organisms or pools of multiple individuals, but recent advances have allowed for sequencing from individuals of small-bodied organisms. Here, we use HMW DNA sequencing with PacBio long reads and TELL-Seq linked reads to assemble and annotate the genome from a single individual feather louse (Brueelia nebulosa) from a European Starling (Sturnus vulgaris). We assembled a genome with a relatively high scaffold N50 (637 kb) and with BUSCO scores (96.1%) comparable to louse genomes assembled from pooled individuals. We annotated a number of genes (10,938) similar to the human louse (Pediculus humanus) genome. Additionally, calling phased variants revealed that the Brueelia genome is more heterozygous (∼1%) then expected for a highly obligate and dispersal-limited parasite. We also assembled and annotated the mitochondrial genome and primary endosymbiont (Sodalis) genome from the individual louse, which showed evidence for heteroplasmy in the mitogenome and a reduced genome size in the endosymbiont compared to its free-living relative. Our study is a valuable demonstration of the capability to obtain high-quality genomes from individual small, nonmodel organisms. Applying this approach to other organisms could greatly increase our understanding of the diversity and evolution of individual genomes.

Taxonomic Revision of Tribe Aleurocanthini Takahashi 1954 stat. rev. Using Consortium Gene Analysis (Mito-Nuclear-Primary Endosymbiont) with the First Evidence for Mitochondrial Recombination in Whitefly (Hemiptera: Aleyrodidae)

The discovery of the Aleurocanthus cf. Aleurocanthus spiniferus (Tea spiny whitefly spiniferus morphotype; previously found in Tokyo) for the first time in Shizuoka Prefecture raised the possibility that this species had invaded Honshu Island, Japan. Unlike the allied species, Aleurocanthus spiniferus and A. camelliae, which have been intercepted from China to Japan, the origin of the current species remains unclear. Despite the status of this species as a minor pest on the ornamental plant, Eurya japonica, the cryptic diversity among the black spiny whitefly is fascinating to be elucidated, specifically how the primary endosymbiont of whiteflies, Portiera aleyrodidarum, coevolved and contributed to the classification of whiteflies. The current study examines the taxonomic status of five species of whiteflies, i.e., A. spiniferus (Quaintance), Aleurocanthus aff. A. camelliae, Aleurocanthus cf. A. spiniferus, A. camelliae Kanmiya and Kasai, Aleurotrachelus camelliae Kuwana, and A. ishigakiensis Takahashi. Using consortium molecular typing targeting mitochondrial DNA (COI and 16S of mitoribosome), the nuclear gene (ITS1), and the ribosomal gene of Portiera, the phylogenetic clustering analysis has been conducted and revealed that the genus Aleurotrachelus sensu lato was clustered together with Aleurocanthini Takahashi, 1954 stat. rev. and reinstated Crenidorsum ishigakiensis comb. nov. due to crescent-shaped scallops being clearly defined. The current study also unveiled several putative species in the A. spiniferus species complex, molecularly. In addition, the recombination event was not detected in Portiera but has been detected in the mtCOI genes of the A. spiniferus cryptic species and the A. woglumi sequences deposited in the NCBI database. The mitochondrial recombination gives an insight into the speciation process among this species complex.

Co-diversification of Camponotus and its Primary Endosymbiont Blochmannia in the USA

Blochmannia is a primary intracellular endosymbiotic bacteria found within the ant tribe Camponotini (Family Formicidae: subfamily Formicinae). Blochmannia is transmitted vertically through maternal host lineages and synthesizes essential amino acids for its host species. The high fidelity of vertical transmission observed means that close co-diversification is expected between Camponotini and their Blochannia hosts. This study expands on past data on USA Camponotini-Blochmannia associations and adds 23 new species to the host COI mitochondrial phylogeny and the 16S ribosomal DNA bacterial phylogeny. Using statistical analyses I found evidence of Blochmannia co-diversification with the host with enhanced species coverage, and one clear host shift is documented.

Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica.

Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286bp and 853,149bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

Plant-mediated rifampicin treatment of Bemisia tabaci disrupts but does not eliminate endosymbionts

Whiteflies are among the most important global insect pests in agriculture; their sustainable control has proven challenging and new methods are needed. Bacterial symbionts of whiteflies are poorly understood potential target of novel whitefly control methods. Whiteflies harbour an obligatory bacterium, Candidatus Portiera aleyrodidarum, and a diverse set of facultative bacterial endosymbionts. Function of facultative microbial community is poorly understood largely due to the difficulty in their selective elimination without removal of the primary endosymbiont. Since the discovery of secondary endosymbionts, antibiotic rifampicin has emerged as the most used tool for their manipulation. Its effectiveness is however much less clear, with contrasting reports on its effects on the endosymbiont community. The present study builds upon most recent method of rifampicin application in whiteflies and evaluates its ability to eliminate obligatory Portiera and two facultative endosymbionts (Rickettsia and Arsenophnus). Our results show that rifampicin reduces but does not eliminate any of the three endosymbionts. Additionally, rifampicin causes direct negative effect on whiteflies, likely by disrupting mitochondria. Taken together, results signify the end of a rifampicin era in whitefly endosymbiont studies. Finally, we propose refinement of current quantification and data analysis methods which yields additional insights in cellular metabolic scaling.

Biochar-Derived Smoke Waters Affect Bactrocera oleae Behavior and Control the Olive Fruit Fly under Field Conditions

Bactrocera oleae is the key pest of olive production. Several attempts have been carried out over time to control it using biological solutions but with results rarely comparable to those obtained with chemical applications. The purpose of this work was to identify and test new compounds from samples of various Smoke Waters (SWs) for their effect on the fly, and given their low impact on the environment. SWs obtained from different feedstocks were used in in vitro and open field applications. SWs were shown to alter B. oleae fitness, acting on its microbiome, particularly on the presence and activity of the primary endosymbiont “Ca. Erwinia dacicola”, and also to affect the behavior of the adult flies, altering the attractiveness of the drupes susceptible to attack. The effects recorded were concentration-dependent and varied among repulsion, up to 87% towards females, indecision, up to 70% towards males, and attraction, comparable to fresh green olives, based on the starting materials. These responses were confirmed in electroantennography trials and during two-years of field trials carried out in South and Central Italy. Gas Chromatography–Mass Spectrometry highlighted the presence of compounds such as guaiacol and hydroquinone as potentially important for the observed activity. Principal Component Analyses confirmed the proximity among SWs obtained from similar feedstocks. In controlled conditions, females appear to be more sensitive to the SW treatments. Field trials have shown how the effects of SWs can lead depression of infestation levels obtainable with other well-known compounds, such as kaolin clay and isopropyl-myristate (repulsive), or pheromones (attractive).

Gut microbiome insights from 16S rRNA analysis of 17-year periodical cicadas (Hemiptera: Magicicada spp.) Broods II, VI, and X

Periodical cicadas (Hemiptera: Magicicada) have coevolved with obligate bacteriome-inhabiting microbial symbionts, yet little is known about gut microbial symbiont composition or differences in composition among allochronic Magicicada broods (year classes) which emerge parapatrically or allopatrically in the eastern United States. Here, 16S rRNA amplicon sequencing was performed to determine gut bacterial community profiles of three periodical broods, including II (Connecticut and Virginia, 2013), VI (North Carolina, 2017), and X (Maryland, 2021, and an early emerging nymph collected in Ohio, 2017). Results showed similarities among all nymphal gut microbiomes and between morphologically distinct 17-year Magicicada, namely Magicicada septendecim (Broods II and VI) and 17-year Magicicada cassini (Brood X) providing evidence of a core microbiome, distinct from the microbiome of burrow soil inhabited by the nymphs. Generally, phyla Bacteroidetes [Bacteroidota] (> 50% relative abundance), Actinobacteria [Actinomycetota], or Proteobacteria [Pseudomonadota] represented the core. Acidobacteria and genera Cupriavidus, Mesorhizobium, and Delftia were prevalent in nymphs but less frequent in adults. The primary obligate endosymbiont, Sulcia (Bacteroidetes), was dominant amongst core genera detected. Chryseobacterium were common in Broods VI and X. Chitinophaga, Arthrobacter, and Renibacterium were common in Brood X, and Pedobacter were common to nymphs of Broods II and VI. Further taxonomic assignment of unclassified Alphaproteobacteria sequencing reads allowed for detection of multiple copies of the Hodgkinia 16S rRNA gene, distinguishable as separate operational taxonomic units present simultaneously. As major emergences of the broods examined here occur at 17-year intervals, this study will provide a valuable comparative baseline in this era of a changing climate.

Microbiome of pear psyllids: A tale about closely related species sharing their endosymbionts.

Psyllids are phloem-feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host-symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure.

Endosymbiotic bacteria of the boar louse Haematopinus apri (Insecta: Phthiraptera: Anoplura).

Insects exclusively feeding on vertebrate blood are usually dependent on symbiotic bacteria for provisioning of B vitamins. Among them, sucking lice are prominent in that their symbiotic bacteria as well as their symbiotic organs exhibit striking diversity. Here we investigated the bacterial diversity associated with the boar louse Haematopinus apri in comparison with the hog louse Haematopinus suis. Amplicon sequencing analysis identified the primary endosymbiont predominantly detected from all populations of H. apri with some minor secondary bacterial associates. Sequencing and phylogenetic analysis of bacterial 16S rRNA gene confirmed that the endosymbionts of the boar louse H. apri, the hog louse H. suis and the cattle louse Haematopinus eurysternus form a distinct clade in the Gammaproteobacteria. The endosymbiont clade of Haematopinus spp. was phylogenetically distinct from the primary endosymbionts of other louse lineages. Fluorescence in situ hybridization visualized the endosymbiont localization within midgut epithelium, ovarial ampulla and posterior oocyte of H. apri, which were substantially the same as the endosymbiont localization previously described in H. suis and H. eurysternus. Mitochondrial haplotype analysis revealed that, although the domestic pig was derived from the wild boar over the past 8,000 years of human history, the populations of H. apri constituted a distinct sister clade to the populations of H. suis. Based on these results, we discussed possible evolutionary trajectories of the boar louse, the hog louse and their endosymbionts in the context of swine domestication. We proposed ‘Candidatus Haematopinicola symbiotica’ for the distinct clade of the endosymbionts of Haematopinus spp.